期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 278, 期 1, 页码 124-130出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M209219200
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资金
- NHLBI NIH HHS [HL-24530] Funding Source: Medline
- NIGMS NIH HHS [GM59427, GM65160, GM 54159] Funding Source: Medline
The present studies mapped the protein kinase A (PKA) phosphorylation site of Galpha(13) and studied the consequences of its phosphorylation. Initial experiments using purified human Galpha(13) and the PKA catalytic subunit established that PKA directly phosphorylates Galpha(13). The location of this phosphorylation site was next investigated with a new synthetic peptide (G(13)SRI(pep)) containing the PKA consensus sequence (Arg-Arg-Pro-Thr(203)) within the switch I region of Galpha(13). G(13)SRI(pep) produced a dose-dependent inhibition of PKA-mediated Ga13 phosphorylation. On the other hand, the Thr-phosphorylated derivative of G(13)SRI(pep) possessed no inhibitory activity, suggesting that Galpha(13) Thr(203) may represent the phosphorylation site. Confirmation of this notion was obtained by showing that the Galpha(13)-T203A mutant (in COS-7 cells) could not be phosphorylated by PKA. Additional studies using co-elution affinity chromatography and co-immunoprecipitation demonstrated that Ga13 phosphorylation stabilized coupling of Galpha(13) with platelet thromboxane A(2) receptors but destabilized coupling of Galpha(13) to its betagamma subunits. In order to determine the functional consequences of this phosphorylation on Ga13 signaling, activation of the Rho pathway was investigated. Specifically, Chinese hamster ovary cells over-expressing human Ga13 wild type (Galpha(13)-WT) or Galpha(13)- T203A mutant were generated and assayed for Rho activation. It was found that 8-bromo-cyclic AMP caused a significant decrease (50%; p < 0.002) of Rho activation in Galpha(13) wild type cells but produced no change of basal Rho activation levels in the mutant (p > 0.4). These results therefore suggest that PKA blocks Rho activation by phosphorylation of Galpha(13) Thr(203).
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